Murine sarcoma virus (MuSV) transformed mouse fibroblasts produce potent in vitro immunosuppressive factors (ISF). The partially purified ISF inhibited phytohemagglutinin (PHA) and concanavalin A (Con A) induced thymocyte proliferation, lipopolysaccharide (LPS)-induced spleen cell proliferation, the in vitro splenic anti-sheep red blood cell plaque forming cell response, and the generation of alloantigen specific cytotoxic T cells. The effect of ISF on thymocyte proliferation was not readily reversible and required only a 4 hr exposure of the thymocytes to ISF in order to inhibit cell proliferation. Although ISF shared several biochemical properties with a MuSV-transformed cell-derived sarcoma growth factor (SGF), e.g., acetic acid solubility, sensitivity to dithiothreitol, the two factors could be resolved by gel filtration on Bio-Gel P-60. Two peaks of ISF activity were found with apparent molecular weights of 12,000 and 8,000.